Inbred squash line KAZA121

ABSTRACT

A novel squash inbred line, designated KAZA121, is disclosed. The invention relates to the seeds of squash inbred line KAZA121, to the plants of squash inbred line KAZA121 and to methods for producing a squash plant by crossing the squash inbred line KAZA121 with itself or another squash line. The invention further relates to methods for producing a squash plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other squash lines derived from the squash inbred line KAZA121.

FIELD OF THE INVENTION

The present invention relates to a new and distinctive squash inbredline, designated “KAZA121”.

BACKGROUND OF THE INVENTION

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In squash, these important traits may include increased fruit number,fruit size and fruit weight, higher seed yield, improved color,resistance to pest, diseases and insects, tolerance to drought and heat,better uniformity, higher nutritional value and better agronomic qualitysuch as sugar levels, small cavity size, flesh color or texture, rindfirmness, strong net, growth rate, high seed germination, seedlingvigor, early fruit maturity, ease of fruit setting, adaptability forsoil and climate conditions.

Practically speaking, all cultivated forms of squash belong to speciesCucurbita pepo L. that is grown for its edible fruit. As a crop, squash,whether summer or winter squash, are grown commercially whereverenvironmental conditions permit the production of an economically viableyield. Both are harvested by hand. Squash usually develop a running vineon the soil but today's summer squash have been developed in the form ofa short compact bush, making them easier to grow in smaller spaces. Onhealthy winter squash plants, there is a canopy of large, reniform andserrated leaves, which may be without lobes or with shallow roundedones. Fruits flesh can be of various shade of yellow. The fruits mayhave a soft or a hard shell with colors from dull to bright orangefleshed or green fleshed. Summer squash show a great variety of shape,with sizes from small to large and colors from uniform to variegated.The flesh can range from white to dark yellow and, contrary to thewinter squash that has a flesh finely grained, bear coarse grains. Inthe United States, the principal fresh market squash growing regions areCalifornia, Florida and Georgia which produce approximately 30,000 acresout of a total annual acreage of more than 57,000 acres (USDA, 2000).Fresh squashes are available in the United States year-round althoughthe greatest supply is from June through October. Summer squash areconsumed immature as table vegetables and winter squash are used whenripe as table vegetable or in pie.

Cucurbita pepo is a member of the family Cucurbitaceae. TheCucurbitaceae is a family of about 90 genera and 700 to 760 species,mostly of the tropics. The family includes pumpkins, squashes, gourds,watermelon, loofah and several weeds. The genus cucurbita, to which thesquash belongs, includes four major species, pepo, mixta, moschata, andmaxima, and one minor species, ficifolia. Cucurbita pepo L. refers towhat is commonly known as the summer squash such as scallop, zucchini,straightneck and crokneck types and winter squash such as acorn andpumpkin. The term squash itself has a rather large meaning. Generally,it can be said that if the plant produce fruits to be harvested in animmature stage, they are called summer squash, and if the fruits are tobe harvested at maturity, they are called winter squash.

Squash is a simple diploid species with twelve pairs of highlydifferentiated chromosomes. The plants are monoecious, with separatefemale and male flowers on the same plant. Usually the first four orfive flowers produced are male, then the next eight or so are female,followed by a few more male flowers. Male flowers have 3-5 erect stamensbunched within the corolla of 5 fused petals. Female flowers have 3spreading stigma lobes and an immature fruit (ovary) below the perianth.The spiny, sticky pollen requires insects for pollination. The primarypollinators are bees, particularly honey bees.

Hybrid vigor has been documented in squashes and hybrids are gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location may be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly used include pedigreeselection, modified pedigree selection, mass selection, recurrentselection and backcross breeding.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars.Nevertheless, it is also suitable for the adjustment and selection ofmorphological character, color characteristics and simply inheritedquantitative characters. Various recurrent selection techniques are usedto improve quantitatively inherited traits controlled by numerous genes.The use of recurrent selection in self-pollinating crops depends on theease of pollination, the frequency of successful hybrids from eachpollination and the number of hybrid offspring from each successfulcross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s) for three or more years.The best lines are candidates for use as parent in new commercialcultivars. Those still deficient in a few traits may be used as parentsto produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and/or to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of squash breeding is to develop new, unique and superiorsquash inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. Another method usedto develop new, unique and superior squash inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental linesfollowed by haploid induction and chromosome doubling that result in thedevelopment of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season. The inbred linesthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large amounts of research monies to develop superior newsquash inbred lines.

The development of commercial squash hybrids requires the development ofhomozygous inbred lines, the crossing of these lines and the evaluationof the hybrid crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine desirable traits from two or more inbred lines or variousbroad-based sources into breeding pools from which inbred lines aredeveloped by selfing and selection of desired phenotypes or through thedihaploid breeding method followed by the selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents that possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families, or hybrid combinations involving individuals ofthese families, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars or as parent of hybrid new cultivars.Similarly, the development of new inbred lines through the dihaploidsystem requires the selection of the best inbreds followed by four tofive years of testing in hybrid combinations in replicated plots.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than to remove one seed from each by hand for thesingle-seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F1 hybrids islost in the next generation (F2). Consequently, seed from F2 hybridvarieties is generally not used for planting stock.

Mutation breeding is another method of introducing new traits intosquash varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in W. R. Fehr, 1993, Principles of Cultivar Development, MacmillanPublishing Co.

Hybrid squash seed is typically produced by a crossing two homozygousinbred lines that possess favorable complementary traits.

Squash is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding squashhybrids that are agronomically sound. The reasons for this goal areobviously to maximize the amount of yield produced on the land. Toaccomplish this goal, the squash breeder must select and develop squashplants that have the traits that result in superior parental lines forproducing superior hybrids.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to the invention, there is provided a novel inbred squash linedesignated KAZA121. This invention thus relates to the seeds of inbredsquash line designated KAZA121, to the plants, or part(s) thereof ofinbred squash line designated KAZA121, to plants or part(s) thereofconsisting essentially of the phenotypic and morphologicalcharacteristics of inbred squash line designated KAZA121, and/or havingall the phenotypic and morphological characteristics of inbred squashline designated KAZA121, and/or having the phenotypic and morphologicalcharacteristics of inbred squash line designated KAZA121 listed in Table1, including but not limited to as determined at the 5% significancelevel when grown in the same environmental conditions. The inventionalso relates to variants, mutants and trivial modifications of the seedor plant of inbred squash line designated KAZA121. Plant parts of theinbred squash line of the present invention are also provided such as,i.e., a scion, a rootstock, a fruit, pollen obtained from the inbredplant and an ovule obtained from the inbred plant.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41 (3) of thePlant Variety Protection Act, i.e., a variety that:

(i) is predominantly derived from inbred squash line designated KAZA121or from a variety that is predominantly derived from inbred squash linedesignated KAZA121, while retaining the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of inbred squash line designated KAZA121;

(ii) is clearly distinguishable from inbred squash line designatedKAZA121; and

(iii) except for differences that result from the act of derivation,conforms to the initial variety in the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of the initial variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of inbred squash line designated KAZA121. Thetissue culture will preferably be capable of regenerating plantsconsisting essentially of the phenotypic and morphologicalcharacteristics of inbred squash line designated KAZA121, and/or havingall the phenotypic and morphological characteristics of inbred squashline designated KAZA121, and/or having the physiological andmorphological characteristics of inbred squash line designated KAZA121.Preferably, the cells of such tissue culture will be embryos,meristematic cells, seeds, stalks, cotyledons, callus, pollen, leaves,anthers, pistils, roots, root tips, flowers, stems and axillary buds.Protoplasts produced from such tissue culture are also included in thepresent invention. The squash shoots, roots and whole plants regeneratedfrom the tissue culture are also part of the invention.

Also included in the invention are methods for producing a squash plantproduced by crossing the inbred squash line designated KAZA121 withitself or another squash line. When crossed with itself, i.e., whencrossed with another inbred squash line designated KAZA121 plant orself-pollinated, inbred squash line designated KAZA121 will be conserved(e.g., as an inbred). When crossed with another, different squash line,an F₁ hybrid seed is produced. F₁ hybrid seeds and plants produced bygrowing said hybrid seeds are included in the present invention. Amethod for producing an F₁ hybrid squash seed comprising crossing aninbred squash line designated KAZA121 plant with a different squashplant and harvesting the resultant hybrid squash seed are also part ofthe invention. The hybrid squash seed produced by the method comprisingcrossing an inbred squash line designated KAZA121 plant with a differentsquash plant and harvesting the resultant hybrid squash seed, areincluded in the invention, as are the hybrid squash plant, or part(s)thereof, fruits and seeds produced by growing said hybrid squash seed.In another embodiment, this invention relates to a method for producingthe inbred squash line KAZA121 from a collection of seeds, thecollection containing both inbred squash line KAZA121 seeds and hybridseeds having inbred squash line KAZA121 as a parental line. Such acollection of seed might be a commercial bag of seeds. Said methodcomprises planting the collection of seeds. When planted, the collectionof seeds will produce inbred squash line KAZA121 plants from inbredsquash line KAZA121 seeds and hybrid plants from hybrid seeds. Theplants having all the physiological and morphological characteristics ofsquash inbred squash line KAZA121 or having a decreased vigor comparedto the other plants grown from the collection of seeds are identified asinbred squash line KAZA121 parent plants. Said decreased vigor is due tothe inbreeding depression effect and can be identified for example by aless vigorous appearance for vegetative and/or reproductivecharacteristics including but not limited to shorter plant height, smallfruit size, or other characteristics As previously mentioned, if theinbred squash line KAZA121 is self-pollinated, the inbred squash lineKAZA121 will be preserved, therefore, the next step is controllingpollination of the inbred parent plants in a manner which preserves thehomozygosity of said inbred squash line KAZA121 parent plant and thefinal step is to harvest the resultant seed.

This invention also relates to methods for producing other inbred squashlines derived from inbred squash line KAZA121 and to the inbred squashlines derived by the use of those methods.

In another aspect, the present invention provides transformed inbredsquash line KAZA121 or part(s) thereof that have been transformed sothat its genetic material contains one or more transgenes, preferablyoperably linked to one or more regulatory elements. Also, the inventionprovides methods for producing a squash plant containing in its geneticmaterial one or more transgenes, preferably operably linked to one ormore regulatory elements, by crossing transformed inbred squash lineKAZA121 with either a second plant of another squash line, or anon-transformed squash plant of the inbred squash line KAZA121, so thatthe genetic material of the progeny that results from the cross containsthe transgene(s), preferably operably linked to one or more regulatoryelements. The invention also provides methods for producing a squashplant that contains in its genetic material one or more transgene(s),wherein the method comprises crossing the inbred squash line KAZA121with a second plant of another squash line which contains one or moretransgene(s) operably linked to one or more regulatory element(s) sothat the genetic material of the progeny that results from the crosscontains the transgene(s) operably linked to one or more regulatoryelement(s). Transgenic squash plants, or part(s) thereof produced by themethods are in the scope of the present invention.

More specifically, the invention comprises methods for producing squashplants or seeds with at least one trait selected from the groupconsisting of male sterile, male fertile, herbicide resistant, insectresistant, disease resistant, water stress tolerant, heat stresstolerant, improved shelf-life, delayed senescence squash plants orseeds. Said methods comprise transforming an inbred squash line KAZA121plant with a nucleic acid molecule that confers, for example, malesterility, male fertility, herbicide resistance, insect resistance,disease resistance, water stress tolerance, heat stress tolerance,improved shelf life or delayed senescence respectively. The transformedsquash plants, or part(s) thereof, or seeds, obtained from the providedmethods, including, for example, a male sterile squash plant, a malefertile squash plant, an herbicide resistant squash plant, an insectresistant squash plant, a disease resistant squash plant, a squash planttolerant to water stress, a squash plant tolerant to heat stress, asquash plant with improved shelf-life, a squash plant with improvedshelf-life and delayed senescence are included in the present invention.Plants may display one or more of the above listed traits. For thepresent invention and the skilled artisan, disease is understood to befungal diseases, viral diseases, bacterial diseases, mycoplasm diseases,or other plant pathogenic diseases and a disease resistant plant willencompass a plant resistant to fungal, viral, bacterial, mycoplasm, andother plant pathogens.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into inbred squash line KAZA121and plants or seeds obtained from such methods. The desired trait(s) maybe, but not exclusively, a single gene, preferably a dominant but also arecessive allele. Preferably, the transferred gene or genes will confersuch traits as male sterility, herbicide resistance, insect resistance,resistance to bacterial, fungal, mycoplasm or viral disease, improvedshelf-life, delayed senescence, tolerance to water stress or heat stressand improved nutritional quality such as altered vitamin content. Thegene or genes may be naturally occurring gene(s) or transgene(s)introduced through genetic engineering techniques. The method forintroducing the desired trait(s) is preferably a backcrossing processmaking use of a series of backcrosses to the inbred squash line KAZA121during which the desired trait(s) is maintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line such as inbred squash line KAZA121 by directtransformation. Rather, the more typical method used by breeders ofordinary skill in the art to incorporate the transgene is to take a linealready carrying the transgene and to use such line as a donor line totransfer the transgene into the newly developed line. The same wouldapply for a naturally occurring trait or one arising from spontaneous orinduced mutations. The backcross breeding process comprises thefollowing steps: (a) crossing inbred squash line KAZA121 plants withplants of another line that comprise the desired trait(s); (b) selectingthe F₁ progeny plants that have the desired trait(s); (c) crossing theselected F₁ progeny plants with inbred squash line KAZA121 plants toproduce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait(s) and physiological andmorphological characteristics of inbred squash line KAZA121 to produceselected backcross progeny plants; and (e) repeating steps (c) and (d)one, two, three, four, five six, seven, eight, nine, or more times insuccession to produce selected, second, third, fourth, fifth, sixth,seventh, eighth, ninth, or higher backcross progeny plants that consistessentially of the phenotypic and morphological characteristics ofinbred squash line KAZA121, and/or have all the phenotypic andmorphological characteristics of inbred squash line KAZA121, and/or havethe desired trait(s) and the physiological and morphologicalcharacteristics of inbred squash line KAZA121 as determined in Table 1,including but not limited to at a 5% significance level when grown inthe same environmental conditions. The squash plants or seeds producedby the methods are also part of the invention. Backcrossing breedingmethods, well-known for one skilled in the art of plant breeding, willbe further developed in subsequent parts of the specification.

Any DNA sequence(s), whether from a different species or from the samespecies that is inserted into the genome using transformation isreferred to herein collectively as “transgenes.” In some embodiments ofthe invention, a transformed variant of KAZA121 may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10transgenes. In another embodiment of the invention, a transformedvariant of the another squash line used as the other parental line maycontain at least one transgene but could contain at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 transgenes.

In an embodiment of this invention is a method of making a backcrossconversion of inbred squash line KAZA121, comprising the steps ofcrossing a plant of squash inbred squash line KAZA121 with a donor plantcomprising a gene, a mutant gene or a transgene conferring a desiredtrait, selecting an F1 progeny plant comprising the gene, the mutantgene or the transgene conferring the desired trait, and backcrossing theselected F1 progeny plant to a plant of inbred squash line KAZA121. Thismethod may further comprise the step of obtaining a molecular markerprofile of inbred squash line KAZA121 and using the molecular markerprofile to select for a progeny plant with the desired trait and themolecular marker profile of inbred squash line KAZA121. In the samemanner, this method may be used to produce an F1 hybrid seed by adding afinal step of crossing the desired trait conversion of inbred squashline KAZA121 with a different squash plant to make F1 hybrid squash seedcomprising a gene, a mutant gene or a transgene conferring the desiredtrait.

In some embodiments of the invention, the number of loci that may bebackcrossed into inbred squash line KAZA121 is at least 1, 2, 3, 4, or5. A single locus may contain several transgenes, such as a transgenefor disease resistance that, in the same expression vector, alsocontains a transgene for herbicide resistance. The gene for herbicideresistance may be used as a selectable marker and/or as a phenotypictrait. A single locus conversion of site specific integration systemallows for the integration of multiple genes at the converted locus.

In a preferred embodiment, the present invention provides methods forincreasing and producing inbred squash line KAZA121 seed, whether bycrossing a first squash parent inbred line plant with a second squashparent inbred line plant and harvesting the resultant squash seed,wherein both said first and second inbred parent squash plant are theinbred squash line KAZA121 or by planting a squash seed of the inbredsquash line KAZA121, growing an inbred squash line KAZA121 plant fromsaid seed, controlling a self-pollination of the plant where the pollenproduced by a grown inbred squash line KAZA121 plant pollinates theovules produced by the very same inbred squash line KAZA121 grown plant,and harvesting the resultant seed.

The invention further provides methods for developing squash plants in asquash breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, molecular markers(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection, and transformation. Seeds,squash plants, and part(s) thereof produced by such breeding methods arealso part of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Adaptability. A plant that has adaptability is a plant able to grow wellin different growing conditions (climate, soils, etc.).

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Earliness. The earliness relates the number of fruits produced from 12to 15 days following the beginning of the harvest: the more fruitsproduced, the more earliness of the plant

Easy to pick fruit. A fruit that is easy to pick is a fruit that easilydetaches from the plant. Once grabbed and twisted, the fruit will breakbetween the peduncle and the stem. For fruits not easy to pick, thepeduncle breaks off the fruits. A fruit that is easy to pick is also afruit that is easily accessible for harvest. When plants have an openplant habit, the fruits are harvested more easily than when the plantshave closed habit.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Extended harvest. An extended harvest is a plant that produces fruitsthroughout the harvest season.

Good Seed Producer. A plant is a good seed producer when it producesnumerous seeds. For squash, a good seed producing plant will produce anaverage of 25 grams of seeds during the harvest season.

Immunity to disease(s) and or insect(s). A squash plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate/Moderate resistance to disease(s) and or insect(s). Asquash plant that restricts the growth and development of specificdisease(s) and or insect(s), but may exhibit a greater range of symptomsor damage compared to high/standard resistant plants. Intermediateresistant plants will usually show less severe symptoms or damage thansusceptible plant varieties when grown under similar environmentalconditions and/or specific disease(s) and or insect(s) pressure, but mayhave heavy damage under heavy pressure. Intermediate resistant squashplants are not immune to the disease(s) and or insect(s).

Mid-Season. The mid-season plant is a plant that is harvestedapproximately 50 days after sowing. An early plant would have 45 daysfrom planting to harvest while a late one will have 55 days.

Open Plant Habit. An open plant habit is a plant where the fruits arevisible without moving the leaves. A plant with closed habit will haveits fruit hidden by leaves that have a high density. An average openplant habit will be between the open and closed habit, and the plantwill have medium leaf density. Whether a plant has open habit or closedhabit is based on the whole of the plant. The more erect the plant, themore compact and therefore the closer the habit. In contrast, when theplant is lodging, sprawling on the ground, it leads to a less compactplant, therefore more “open”.

Plant Habit. A plant can be an upright plant (also called erect) or canbe lodging on the ground. It can also be pendant.

Squash Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe squash at harvest.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell. As used herein, the teem “plant cell” includes plant cellswhether isolated, in tissue culture, or incorporated in a plant or plantpart.

Plant part. As used herein, the term “plant part” includes any part ofthe plant including but not limited to leaves, heads, stems, roots,seed, embryos, pollen, ovules, flowers, root tips, anthers, tissue,cells, axillary buds, rootstock, scion, fruits and the like.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Ribs. The ribs on the fruit may be prominent, inconspicuous ornonexistent. They refer to the ridges along the fruit mostly near thepeduncle.

High/standard resistance to disease(s) and or insect(s). A squash plantthat restricts highly the growth and development of specific disease(s)and or insect(s) under normal disease(s) and or insect(s) attackpressure when compared to susceptible plants. These squash plants canexhibit some symptoms or damage under heavy disease(s) and or insect(s)pressure. Resistant squash plants are not immune to the disease(s) andor insect(s).

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

Susceptible to disease(s) and or insect(s). A squash plant that issusceptible to disease(s) and or insect(s) is defined as a squash plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A squash plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Small plant. A small plant has short internodes with petiole lengths ofapproximately 40 cm and a plant height of 40 to 60 cm. It depends on howthe plant spreads out horizontally or vertically.

Large plant. A large plant has long internodes with a plant height of 75cm and above. It depends on how the plant spreads out horizontally orvertically.

DETAILED DESCRIPTION OF THE INVENTION

Inbred squash line KAZA121 has superior characteristics and wasdeveloped from the cross No. AD007 of plot 99 854.1*853.1/1 made inspring at the Clause Tezier Research Station in Maninet, France

Individual plants were selected among the AD007 cross progenies andself-pollinated in March. The plants obtained therefrom were selectedand self pollinated in September. This breeding scheme was repeated overfour years, two generations per year. This pedigree selection was aimedfor vigorous, erected plant, black fruit and high production andresistance for Powdery Mildew and Zucchini Yellow Mosaic Virus.

In April of the fourth year, the plant AD007.12.14.3.4.9.3.7.5 wasfinally selected and bulked. The line was designated as inbred lineKAZA121.

KAZA121 is similar to Black Magic with black fruit color and goodadaptation to spring and counter season conditions. Nevertheless,KAZA121 is more vigorous with a better plant habit, the plants beingmore erected and more open. The fruit quality of KAZA121 is also muchbetter, the fruits being longer and more shinny. The fruit production isalso higher for KAZA121 when compared to Black Magic

Inbred squash KAZA121 is a black zucchini summer squash with superiorcharacteristics. It provides an excellent parental line in crosses forproducing first generation hybrid squash.

KAZA121 is particularly suitable to produce dark green to black zucchinihybrids bests adapted to spring and counter season in Mexico, Floridaand Europe (Spain, Italy). However it's superior characteristics andresistance make it usable to produce a wide range of varieties in manyother areas such as open field crop for California or Eastern US. Atmarketable maturity, fruits are fully black, very shiny, cylindrical andvery uniform, flower scared is very small. Yield is very high andearliness is outstanding. The inbred plants is also very good, beingupright and open with vigor characteristics suitable for counter season.

KAZA121 is resistant to Zucchini Yellow Mosaic Virus (ZYMV) and PowderyMildew (PM) and tolerant to Watermelon Mosaic Virus (WMV).

KAZA121 will be used to produce counter season high yielding hybrids,with plants having an open habit, with easy to pick fruits having ablack color for Mexico and California to dark green for Eastern US andhaving resistance to Zucchini Yellow Mosaic Virus and Powdery Mildew andtolerance to Watermelon Mosaic.

During the development of the line, first cross were made for thepurpose of combining high yield, good fruit quality and good vigorsuitable for counter season.

Some of the selection criteria used for various generations includeZucchini Yellow Mosaic Virus and Powdery Mildew resistance because suchresistances may be of great value in new squash varieties development.PM and ZYMV were evaluated in Maninet (Valence. French Research station)in parallel after each generation to continue only the resistant to bothpathogen seeds lot.

The inbred was evaluated further as a line and in numerous crosses byManinet, Valence. French Research station. The inbred has proven to havea good combining ability in hybrid combinations.

Squash inbred line KAZA121 has shown uniformity and stability for thetraits, as described in the following Variety Description Information.It has been self-pollinated a sufficient number of generations withcareful attention to uniformity of plant type. The cultivar has beenincreased with continued observation for uniformity. Several trialsshowed very good adaptation of the plant in counter-season growingconditions with a very good productivity in the fall and wintergreenhouses of Almeria, Spain No variant traits have been observed orare expected for agronomically important traits in squash inbred lineKAZA121.

Squash inbred line KAZA121 has the following morphologic and othercharacteristics (based primarily on data collected at Salinas, Calif.).

TABLE 1 VARIETY DESCRIPTION INFORMATION SPECIES: pepo KIND: squash TYPE:summer COTYLEDONS Shape: elliptic Green color of cotyledons: dark PLANTGrowth habit: bush Branching: absent Attitude of petiole: horizontalLEAVES Leaf blade size: medium Leaf blade incisions: deep Intensity ofgreen color of upper surface: dark Silvery patches: present Relativearea covered by silvery patches: large Leaf surface: blistered PETIOLENumber of prickles: few FRUIT (at market maturity): Fruit general shape:cylindrical Apex: flattened Base: rounded Ribs: present Protrusion ofribs: medium Color of ribs compared to main color of skin: same Grooves:none Stripes: absent Fruit surface: smooth Warts: absent Size of flowerscar: very small Main color of skin: green Intensity of green color ofskin: very dark Fruit length: medium Fruit ratio length/maximumdiameter: medium Dots: present Size of main dots: very small Length ofpedoncule: short Color of pedoncule: green Intensity of green color ofpedoncule: medium Molting of pedoncule: present RIND Rind color: blackgreen SEEDS: Size: medium Shape: elliptic Hull: present Appearance ofhull: fully developed Length: 12.7 mm Width: 7.1 mm Thickness: 2.8 mmFace surface: smooth Color: cream Number of seeds per fruit: 106 Weightof 100 seeds: 10.2 gm DISEASE RESISTANCE Rating (1 = susceptible-2 =tolerant-3 = resistant) Cucumber Mosaic Virus (CMV): 1 Zucchini YellowMosaic Virus (ZYMV): 3 Watermelon Mosaic Virus (WMV): 2 Papaya Ring SpotVirus (PRSV): 1 Squash Leaf Curl Virus (SLCV): 1 Powdery mildew: 3

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a squash plantby crossing a first parent squash plant with a second parent squashplant wherein either the first or second parent squash plant is aninbred squash plant of inbred squash line KAZA121. Further, both firstand second parent squash plants can come from the inbred squash lineKAZA121. When self pollinated, or crossed with another squash inbredline KAZA121 plant, the inbred squash plant KAZA121 will be stable,while when crossed with another, different squash line, an F₁ hybridseed is produced. Such methods of hybridization and self-pollination arewell known to those skilled in the art of breeding. See, for example,Fehr and Hadley, eds., Chapter 17: 273-284, American Society of Agronomyand Crop Science Society of America, Publishers.

An inbred squash line has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.An inbred line can also be produced though the dihaploid system whichinvolves doubling the chromosomes from a haploid plant or embryo thusresulting in an inbred line that is genetically stable (homozygous) andcan be reproduced without altering the inbred line: Haploid plants couldbe obtained from haploid embryos that might be produced frommicrospores, pollen, anther cultures or ovary cultures. The haploidembryos may then be doubled by chemical treatments such as by colchicineor be doubled autonomously. The haploid embryos may also be grown intohaploid plants and treated to induce the chromosome doubling. In eithercases, fertile homozygous plants are obtained. A hybrid variety isclassically created through the fertilization of an ovule from an inbredparental line by the pollen of another, different inbred parental line.Due to the homozygous state of the inbred line, the produced gametescarry a copy of each parental chromosome. As both the ovule and thepollen bring a copy of the arrangement and organization of the genespresent in the parental lines, the genome of each parental line ispresent in the resulting F1 hybrid, theoretically in the arrangement andorganization created by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross is stable. The F1 hybrid is then a combination ofphenotypic characteristics issued from two arrangement and organizationof genes, both created by one skilled in the art through the breedingprocess.

Still further, this invention is also directed to methods for producingan inbred squash line KAZA121-derived squash plant by crossing inbredsquash line KAZA121 with a second squash plant and growing the progenyseed, and repeating the crossing and growing steps with the inbredsquash line KAZA121-derived plant from 0 to 7 times. Thus, any suchmethods using the inbred squash line KAZA121 are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using inbred squash line KAZA121 as a parentare within the scope of this invention, including plants derived frominbred squash line KAZA121. Such plants might exhibit additional anddesired characteristics or traits such as high seed yield, high seedgermination, seedling vigor, early fruit maturity, high fruit yield,ease of fruit setting, disease tolerance or resistance, and adaptabilityfor soil and climate conditions. Consumer-driven traits, such as apreference for a given fruit size, shape, color, texture, and taste,especially non-pungency (low capsaicinoid content) or increased vitamincontent, are other traits that may be incorporated into new squashplants developed by this invention.

Particularly desirable traits that may be incorporated by this inventionare improved resistance to different viral, fungal, and bacterialpathogens such as resistance to Powdery Mildew (PM), Zucchini YellowMosaic virus (ZYMV), Cucumber Mosaic Virus (CMV), Watermelon MosaicVirus (WMV), Squash Leaf Curl Virus (SLCV), Papaya Ringspot Virus (PRSV)

Improved resistance to insect pests is another desirable trait that maybe incorporated into new squash plants developed by this invention.Insect pests affecting the various species of squash include theEuropean corn borer, corn earworm, aphids, flea beetles, whiteflies, andmites (Midwest Vegetable Production Guide for Commercial Growers, 2003).

Advantageously, the cultivar is used in crosses with other, different,cultivars to produce first generation (F₁) squash hybrid seeds andplants with superior characteristics.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which squash plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, heads, stems, roots, anthers, pistils, root tips, leaves,meristematic cells, axillary buds and the like. As it is well known inthe art, tissue culture of squash can be used for the in vitroregeneration of squash plants. Tissues cultures of various tissues ofsquash and regeneration of plants therefrom are well known andpublished. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants as described in Kintzios et al.,Acta Horticulturae. 1998, 461, 427-432, Chee-PP. Hort Science, 1992, 27:1, 59-60, Chee-PP. Plant Cell Report 1991, 9: 11, 620-622, Juretic etal., Plant Cell Report. 1991, 9: 11, 623-626, Rakoczy et al., Plant CellTissue and Organ Culture 1989, 18: 2, 191-194, Hegazi H H. ArabUniversity Journal of Agricultural Science. 1999, 7: 2, 507-520, alsoSchroder, Bot. Gaz. 129:374-376 (1968) reported the production ofembryogenic tissue from pericarp tissues of squash. Jelaska, Planta103:278-280 (1972) and Acta Bot. Croat. 32: 81-94 (1973) reportedsomatic embryogenesis in hypocotyl and cotyledon-derived callus ofpumpkins and demonstrated that embryos could develop into normal plants.Pink et al., Sci. Hortic. 24:107-114 (1984) reported a rapid propagationmethod for pumpkin through apical meristem culture. See also Toppi etal., Plant Cell Tissue and Organ Culture 51:2 89-93 (1997) and U.S. Pat.No. 5,677,157 filed in 1994. Thus, it is clear from the literature thatthe state of the art is such that these methods of obtaining plants areroutinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce squash plants having the physiological andmorphological characteristics of inbred squash line KAZA121.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary budsand the like. Means for preparing and maintaining plant tissue cultureare well known in the art. By way of example, a tissue culturecomprising organs has been used to produce regenerated plants. U.S. Pat.Nos. 5,959,185, 5,973,234, and 5,977,445 describe certain techniques,the disclosures of which are incorporated herein by reference.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line. An embodiment of the presentinvention comprises at least one transformation event in inbred squashline KAZA121.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedsquash plants using transformation methods as described below toincorporate transgenes into the genetic material of the squash plant(s).

Expression Vectors for Squash Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford, et al.,Plant Physiol., 86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86(1987); Svab, et al., Plant Mol. Biol., 14:197 (1990); Hille, et al.,Plant Mol. Biol., 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate or bromoxynil(Comai, et al., Nature, 317:741-744 (1985); Gordon-Kamm, et al., PlantCell, 2:603-618 (1990); and Stalker, et al., Science, 242:419-423(1988)).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells (Chalfie,et al., Science, 263:802 (1994)). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Squash Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in squash. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in squash. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See, Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett, et al., Proc. Natl. Acad. Sci. USA,90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Gatz, et al., Mol. Gen. Genetics,243:32-38 (1994)), or Tet repressor from Tn10 (Gatz, et al., Mol. Gen.Genetics, 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena,et al., Proc. Natl. Acad. Sci. USA, 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in squash or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in squash.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell 2, 163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genetics, 231:276-285 (1992) and Atanassova, et al., Plant Journal,2 (3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See, PCT Application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in squash.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in squash. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., Proc. Natl. Acad. Sci. USA82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson, et al., EMBO J., 4(11):2723-2729(1985) and Timko, et al., Nature, 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell, et al., Mol.Gen. Genetics, 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 or a microspore-preferred promoter such as that from apg(Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine during protein synthesis and processing where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., etal., “Structure and Organization of Two Divergent Alpha-Amylase Genesfrom Barley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., Proc. Natl. Acad. Sci., 88:834 (1991); Gould,et al., J. Cell. Biol., 108:1657 (1989); Creissen, et al., Plant J.,2:129 (1991); Kalderon, et al., Cell, 39:499-509 (1984); Steifel, etal., “Expression of a maize cell wall hydroxyproline-rich glycoproteingene in early leaf and root vascular differentiation,” Plant Cell,2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a squash plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Methods in Plant Molecular Biology and Biotechnology,Glick and Thompson Eds., CRC Press, Inc., Boca Raton, 269:284 (1993).Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defences are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example, Jones, et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); Mindrinos, et al., Cell, 78:1089 (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modelled thereon. See, for example, Geiser, et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

C. A lectin. See, for example, Van Damme, et al., Plant Molec. Biol.,24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See, PCT Application US93/06487 which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Molec. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani, etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosure of Pratt, et al., Biochem. Biophys. Res. Comm., 163:1243(1989) (an allostatin is identified in Diploptera puntata). See also,U.S. Pat. No. 5,266,317 to Tomalski, et al., which discloses genesencoding insect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule, forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, PCTApplication WO 93/02197 (Scott, et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also, Kramer, et al., InsectBiochem. Molec. Biol., 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck, et al.,Plant Molec. Biol., 21:673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Molec. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See, PCT Application WO 95/16776, whichdiscloses peptide derivatives of tachyplesin which inhibit fungal plantpathogens, and PCT Application WO 95/18855, which teaches syntheticantimicrobial peptides that confer disease resistance.

M. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci, 89:43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilising plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/Technology,10:1436 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/Technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

S. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., “Plant diseaseresistance. Grand unification system theory in sight,” Current Biology,5 (2) (1995).

T. Antifungal genes. See, Cornelissen and Melchers, “Strategies forControl of Fungal Diseases with Transgenic. Plants,” Plant Physiol.,101:709-712 (1993); and Bushnell, et al., “Genetic Engineering ofDisease Resistance in Cereal,” Can. J. of Plant Path., 20(2):137-149(1998).

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988), and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSPS which can confer glyphosate resistance. ADNA molecule encoding a mutant aroA gene can be obtained under ATCCAccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061 to Comai. European PatentApplication No. 0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374to Goodman, et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. See also, Russel, D. R., et al., Plant Cell Report,12:3 165-169 (1993). The nucleotide sequence of aphosphinothricin-acetyl-transferase (PAT) gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/Technology, 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori, et al., “AnAcetohydroxy acid synthase mutant reveals a single site involved inmultiple herbicide resistance,” Mol. Gen. Genet., 246:419-425 (1995).Other genes that confer tolerance to herbicides include a gene encodinga chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochromeP450 oxidoreductase (Shiota, et al., “Herbicide-resistant Tobacco PlantsExpressing the Fused Enzyme between Rat Cytochrome P4501A1 (CYP1A1) andYeast NADPH-Cytochrome P450 Oxidoreductase,” Plant Physiol., 106:17(1994)), genes for glutathione reductase and superoxide dismutase (Aono,et al., “Paraquat tolerance of transgenic Nicotiana tabacum withenhanced activities of glutathione reductase and superoxide dismutase,”Plant Cell Physiol., 36:1687 (1995)), and genes for variousphosphotransferases (Datta, et al., “Herbicide-resistant Indica riceplants from IRRI breeding line IR72 after PEG-mediated transformation ofprotoplants,” Plant Mol. Biol., 20:619 (1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the squash, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae, 521, 101-109 (2000).

C. Increased sweetness of the squash by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology, 10: 5, 561-564(1992).

D. Delayed senescence or browning by transferring a gene or acting onthe transcription of a gene involved in the plant senescence. See Wanget al. In Plant Mol. Bio, 52:1223-1235 (2003) on the role of thedeoxyhypusine synthase in the senescence. See also U.S. Pat. No.6,538,182 issued Mar. 25, 2003.

Methods for Squash Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in-vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch, et al., Science,227:1229 (1985); Diant, et al., Molecular Breeding, 3:1, 75-86 (1997).A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteriawhich genetically transform plant cells. The Ti and Ri plasmids of A.tumefaciens and A. rhizogenes, respectively, carry genes responsible forgenetic transformation of the plant. See, for example, Kado, C. I.,Crit. Rev. Plant Sci., 10:1 (1991). Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by Gruber, et al., supra, Miki, et al., supra, and Moloney, etal., Plant Cell Reports, 8:238 (1989). See also, U.S. Pat. No. 5,591,616issued Jan. 7, 1997.

B. Direct Gene Transfer—Despite the fact the host range forAgrobacterium-mediated transformation is broad, some cereal or vegetablecrop species and gymnospeinis have generally been recalcitrant to thismode of gene transfer, even though some success has been achieved inrice and corn. Hiei, et al., The Plant Journal, 6:271-282 (1994) andU.S. Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation. A generally applicable method of plant transformation ismicroprojectile-mediated transformation where DNA is carried on thesurface of microprojectiles measuring 1 to 4 microns. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al., Pl. Cell. Rep., 12, 165-169 (3 Jan. 1993); Aragao, F. J. L., etal., Plant Mol. Biol., 20, 357-359 (2 Oct. 1992); Aragao, Theor. Appl.Genet., 93:142-150 (1996); Kim, J.; Minamikawa, T., Plant Science,117:131-138 (1996); Sanford, et al., Part. Sci. Technol., 5:27 (1987);Sanford, J. C., Trends Biotech., 6:299 (1988); Klein, et al., Bio/Tech.,6:559-563 (1988); Sanford, J. C., Physiol Plant, 7:206 (1990); Klein, etal., Biotechnology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985); Christou, et al., Proc Natl. Acad. Sci. USA, 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. Hain, etal., Mol. Gen. Genet., 199:161 (1985) and Draper, et al., Plant CellPhysiol., 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described Saker, M. and Kuhne, T., BiologiaPlantarum, 40(4):507-514 (1997/98); D'Halluin, et al., Plant Cell,4:1495-1505 (1992); and Spencer, et al., Plant Mol. Biol., 24:51-61(1994)).

Following transformation of squash target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed with another (non-transformed or transformed) inbredline in order to produce a new transgenic inbred line. Alternatively, agenetic trait that has been engineered into a particular squash lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into a inbred line or lines that do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross or the process of backcrossing depending on the context.

Backcrossing

When the term inbred squash plant is used in the context of the presentinvention, this also includes inbred squash plant where one or moredesired traits has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the inbred. The term “backcrossing”as used herein refers to the repeated crossing of a hybrid progeny backto the recurrent parent, i.e., backcrossing one, two, three, four, five,six, seven, eight, nine, or more times to the recurrent parent. Theparental squash plant which contributes the gene for the desiredcharacteristic is teemed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental squash plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol.

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a squash plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, generally determined at a 5% significance levelwhen grown in the same environmental condition, in addition to the geneor genes transferred from the nonrecurrent parent. It has to be notedthat some, one, two, three, or more, self-pollination and growing ofpopulation might be included between two successive backcrosses. Indeed,an appropriate selection in the population produced by theself-pollination, i.e., selection for the desired trait andphysiological and morphological characteristics of the recurrent parentmight be equivalent to one, two or even three, additional backcrosses ina continuous series without rigorous selection, saving time, money andeffort to the breeder. A non limiting example of such a protocol wouldbe the following: (a) the first generation F₁ produced by the cross ofthe recurrent parent A by the donor parent B is backcrossed to parent A;(b) selection is practiced for the plants having the desired trait ofparent B; (c) selected plants are self-pollinated to produce apopulation of plants where selection is practiced for the plants havingthe desired trait of parent B and the physiological and morphologicalcharacteristics of parent A; (d) the selected plants are backcrossedone, two, three, four, five, six, seven, eight, nine, or more times toparent A to produce selected backcross progeny plants comprising thedesired trait of parent B and the physiological and morphologicalcharacteristics of parent A. Step (c) may or may not be repeated andincluded between the backcrosses of step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a gene or genes of the recurrent inbred ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicalimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a single geneand dominant allele, multiple genes and recessive allele(s) may also betransferred and therefore, backcross breeding is by no means restrictedto character(s) governed by one or a few genes. In fact the number ofgenes might be less important than the identification of thecharacter(s) in the segregating population. In this instance it may thenbe necessary to introduce a test of the progeny to determine if thedesired characteristic(s) has been successfully transferred. Such testsencompass visual inspection, simple crossing but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele requiresselfing the progeny to determine which plant carries the recessiveallele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic. An example of a gene controlling resistance to tomatospotted wilt virus in squash can be found in a publication of Boiteux LS and de Avila A C, Inheritance of a resistance specific to tomatospotted wilt tospovirus in Capsicum chinense ‘PI 159236’, Euphytica 75,139-142 (1994). These genes are generally inherited through the nucleus.Some other single gene traits are described in U.S. Pat. Nos. 5,777,196,5,948,957, and 5,969,212, the disclosures of which are specificallyhereby incorporated by reference.

In 1981 the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred corn line development in the United States,according to, Hallauer, A. R., et al., “Corn Breeding,” Corn and CornImprovement, No. 18, pp. 463-481 (1988).

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,Principles of Plant Breeding, published by John Wiley & Sons, Inc.) Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a variety with exactly the adaptation, yielding ability, andquality characteristics of the recurrent parent but superior to thatparent in the particular characteristic(s) for which the improvementprogram was undertaken. Therefore, this method provides the plantbreeder with a high degree of genetic control of his work.

The backcross method is scientifically exact because the morphologicaland agricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method,” Jour. Amer. Soc. Agron.,22:289-244 (1930)).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart’ wheat and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in ‘CaliforniaCommon’ alfalfa to create ‘Caliverde’. This new ‘Caliverde’ varietyproduced through the backcross process is indistinguishable from‘California Common’ except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics, and simply inherited quantitative characters,such as earliness, plant height, and seed size and shape. In thisregard, a medium grain type variety, ‘Calady’, has been produced byJones and Davis. As dealing with quantitative characteristics, theyselected the donor parent with the view of sacrificing some of theintensity of the character for which it was chosen, i.e., grain size.‘Lady Wright’, a long grain variety was used as the donor parent and‘Coloro’, a short grain variety as the recurrent parent. After fourbackcrosses, the medium grain type variety ‘Calady’ was produced.

DEPOSIT INFORMATION

A deposit of the squash seed of this invention is maintained by ClauseRue Louis Saillant, Z. I. La Motte, 26800 Portes les Valence, France. Inaddition, a sample of the squash seed of this invention has beendeposited with the National Collections of Industrial, Food and MarineBacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY,United Kingdom, on May 26, 2011.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited squash line KAZA121(deposited as NCIMB Accession No. 41843):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. Upon granting of the patent the strain will be available to thepublic under conditions specified in 37 CFR 1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the enforceable lifeof the patent, whichever is longer;

4. The viability of the biological material at the time of deposit willbe tested; and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

TABLES

In Table 2 the traits and characteristics of inbred KAZA121 are givencompared to Black Magic. The data collected are presented for keycharacteristics and traits. The field tests are experimental trialsunder supervision of the applicant.

The data was collected during one growing season from field locations inAlmeria, Spain.

Table 2:

The first column shows the plant vigor with a rating from 1 to 5, 1being very weak, 5 being very strong.

The second column shows the plant habit with a rating from 1 to 5, 1being very close, 5 being very open.

The third column shows the fruit color with a rating from 1 to 5, 1being very light green, 5 being very dark green.

The fourth column shows the fruit brightness with a rating from 1 to 5,1 being very dull, 5 being very shinny

The fifth column shows the fruit length with a rating from 1 to 5, 1 isbeing very short, 5 being very long

The six column shows the earliness with an average of fruit per plantafter 12 days of harvest;

TABLE 2 Characteristic Comparisons for Field Trials in Spain, in Almeriaarea. Open/ Earliness Plant close Fruit Fruit (total Name vigor plantcolor Brightness length fruit/pl) KAZA121 4 4 5 4 4 5 Black Magic 3 2 52 3 2.2

In tables 3 and 4 that follow, the traits and characteristics of hybridcontaining inbred squash KAZA121 as a parent are given compared to otherhybrids. The data collected are presented for key characteristics andtraits. The field tests are experimental trials under supervision of theapplicant. The hybrid TOSCA, CORA and CRONOS are hybrid checks whileVN172, VL502, VK119 and VM936 are hybrids having KAZ121 as a parent.

The data was collected during one growing season from field locations inAlmeria, Spain.

The first column shows the plant vigor with a rating from 1 to 5, 1being very weak, 5 being very strong.

The second column shows the plant habit with a rating from 1 to 5, 1being very close, 5 being very open.

The third column shows the fruit color with a rating from 1 to 5, 1being very light green, 5 being very dark green.

The fourth column shows the fruit brightness with a rating from 1 to 5,1 being very dull, 5 being very shinny.

The fifth column shows the fruit length with a rating from 1 to 5, 1 isbeing very short, 5 being very long.

The sixth column shows the yield total with an average of fruit perplant after 12 days of harvest.

TABLE 3 Yield Plant Open/close Fruit Fruit (total Name vigor plant colorBrightness length fruit/pl) TOSCA 5 4 3 4 4 4.57 CRONOS 4 5 5 2 5 3.57VN172 5 4 5 5 5 4.85 VL502 4 4 4 4 4 4.37 VM936 5 4 4 4 3 5.24

TABLE 4 Yield Plant Open/close Fruit Fruit (total Name vigor plant colorBrightness length fruit/pl) CORA 4 4 3 4 4 12 CRONOS 3, 5 4 5 3 5 10.3VN172 4 4 5 5 4 16.6 VL502 4 5 4 4 3 14.3 VK119 4 4 5 4 2 11

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of inbred squash line designated KAZA121,wherein a representative sample of seed of said line was deposited underNCIMB Accession No.
 41843. 2. A squash plant, or a part thereof,produced by growing the seed of claim
 1. 3. A squash plant, or a partthereof, having all the physiological and morphological characteristicsof inbred line KAZA121, wherein a representative sample of seed of saidline was deposited under NCIMB Accession No.
 41843. 4. A tissue cultureof regenerable cells produced from the plant of claim 2, wherein saidcells of the tissue culture are produced from a plant part selected fromthe group consisting of embryos, meristematic cells, leaves, pollen,callus, root, root tips, stems, anther, pistils, axillary buds, flowers,cotyledons, stalks and seeds.
 5. A squash plant regenerated from thetissue culture of claim 4, said plant having all the morphological andphysiological characteristics of inbred line KAZA121, wherein arepresentative sample of seed of said line was deposited under NCIMBAccession No.
 41843. 6. A method for producing a hybrid squash seed,said method comprising crossing a first parent squash plant with asecond parent squash plant and harvesting the resultant hybrid squashseed, wherein said first parent squash plant or second parent squashplant is the squash plant of claim
 2. 7. A hybrid squash seed producedby the method of claim
 6. 8. A method for producing inbred squash lineKAZA121, wherein a representative sample of seed of said line wasdeposited under NCIMB Accession No. 41843, wherein the method comprises:a) planting a collection of seed comprising seed of a hybrid, one ofwhose parents is inbred line KAZA121, said collection also comprisingseed of said inbred; b) growing plants from said collection of seed; c)identifying the plants having all the physiological and morphologicalcharacteristics of inbred squash line KAZA121 as inbred parent plants;d) controlling pollination of said inbred parent plants in a mannerwhich preserves the homozygosity of said inbred parent plant; and e)harvesting the resultant seed.
 9. The method of claim 8 wherein step (c)comprises identifying plants with decreased vigor compared to the otherplants grown from the collection of seeds.
 10. A method for producing anherbicide resistant plant, said method comprising transforming thesquash plant of claim 2 with a transgene that confers herbicideresistance to an herbicide selected from the group consisting ofimidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine, and benzonitrile.
 11. An herbicideresistant squash plant, or a part thereof, produced by the method ofclaim
 10. 12. A method for producing an insect resistant squash plant,said method comprising transforming the squash plant of claim 2 with atransgene that confers insect resistance.
 13. An insect resistant squashplant, or a part thereof, produced by the method of claim
 12. 14. Amethod for producing a disease resistant squash plant, said methodcomprising transforming the squash plant of claim 2 with a transgenethat confers disease resistance.
 15. A disease resistant squash plant,or a part thereof, produced by the method of claim
 14. 16. A method ofintroducing a desired trait into squash inbred line KAZA121, said methodcomprising: (a) crossing a squash inbred line KAZA121 plant grown fromsquash inbred line KAZA121 seed, wherein a representative sample of seedhas been deposited under NCIMB No. 41843, with another squash plant thatcomprises a desired trait to produce F₁ progeny plants, wherein thedesired trait is selected from the group consisting of insectresistance, disease resistance, water stress tolerance, heat tolerance,improved shelf life, delayed senescence, and improved nutritionalquality; (b) selecting one or more progeny plants that have the desiredtrait to produce selected progeny plants; (c) crossing the selectedprogeny plants with the squash inbred line KAZA121 plants to producebackcross progeny plants; (d) selecting for backcross progeny plantsthat have the desired trait and all the physiological and morphologicalcharacteristics of squash inbred line KAZA121 listed in Table 1 toproduce selected backcross progeny plants; and (e) repeating steps (c)and (d) three or more times in succession to produce selected fourth orhigher backcross progeny plants that comprise the desired trait and allthe physiological and morphological characteristics of squash inbredline KAZA121 listed in Table
 1. 17. A squash plant produced by themethod of claim 16, wherein the plant has the desired trait and all thephysiological and morphological characteristics of squash inbred lineKAZA121 listed in Table
 1. 18. A method for producing squash inbred lineKAZA121 seed comprising crossing a first parent squash plant with asecond parent squash plant and harvesting the resultant squash seed,wherein both said first and second squash plants are the squash plant ofclaim 2 or
 3. 19. The squash plant of claim 17, wherein the desiredtrait is herbicide resistance and the resistance is conferred to anherbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine, andbenzonitrile.
 20. The squash plant of claim 17, wherein the desiredtrait is insect resistance and the insect resistance is conferred by atransgene encoding a Bacillus thuringiensis endotoxin.
 21. The squashplant of claim 17, wherein the desired trait is selected from the groupconsisting of insect resistance, disease resistance, water stresstolerance, heat tolerance, improved shelf life, delayed senescence, andimproved nutritional quality.
 22. A method for producing a hybrid squashseed comprising crossing a first parent squash plant with a secondparent squash plant and harvesting the resultant hybrid squash seed,wherein said first parent squash plant or second parent squash plant isthe squash plant of claim
 17. 23. A hybrid squash plant produced by themethod of claim
 22. 24. A hybrid squash plant produced by the hybridsquash seed of claim
 7. 25. A hybrid squash fruit produced by the hybridsquash plant of claim
 24. 26. A method for producing inbred squash lineKAZA121 seed, wherein a representative sample of seed of said line wasdeposited under NCIMB Accession No. 41843, wherein the method comprises:a) planting the inbred squash seed of claim 1; b) growing a plant fromsaid seed; c) controlling pollination in a manner that the pollenproduced by the grown plant pollinates the ovules produced by the grownplant; and d) harvesting the resultant seed.